Downregulation of Endocannabinoid Signaling in the
Hippocampus Following Chronic Unpredictable Stress
Matthew N Hill1,4, Sachin Patel2,4, Erica J Carrier2, David J Rademacher2, Brandi K Ormerod1,3,
Cecilia J Hillard*,2and Boris B Gorzalka*,1
1Department of Psychology, University of British Columbia, Vancouver, BC, Canada V6T 1Z4;2Department of Pharmacology and Toxicology,
Medical College of Wisconsin, Milwaukee, USA;3Neuroscience Program, University of British Columbia, Vancouver, BC, Canada V6T 1Z4
Deficits in cognitive functioning and flexibility are seen following both chronic stress and modulation of endogenous cannabinoid (eCB)
signaling. Here, we investigated whether alterations in eCB signaling might contribute to the cognitive impairments induced by chronic
stress. Chronic stress impaired reversal learning and induced perseveratory behavior in the Morris water maze without significant effect
on task acquisition. These cognitive impairments were reversed by exogenous cannabinoid administration, suggesting deficient eCB
signaling underlies these phenomena. In line with this hypothesis, chronic stress downregulated CB1receptor expression and significantly
reduced the content of the endocannabinoid 2-arachidonylglycerol within the hippocampus. CB1 receptor density and
2-arachidonylglycerol content were unaffected in the limbic forebrain. These data suggest that stress-induced downregulation of
hippocampal eCB signaling contributes to problems in behavioral flexibility and could play a role in the development of perseveratory and
ruminatory behaviors in stress-related neuropsychiatric disorders.
Neuropsychopharmacology (2005) 30, 508–515, advance online publication, 3 November 2004; doi:10.1038/sj.npp.1300601
Keywords: chronic stress; endocannabinoid; perseveration; hippocampus; CB1; 2-arachidonylglycerol
Stress is a phenomenon during which an organism initiates
a repertoire of physiological responses in an attempt to
maintain homeostasis in the face of aversive stimuli. In the
short term, these responses include mobilization of various
autonomic and behavioral responses that are beneficial to
the organism. However, chronic hyperactivity of neural
pathways subserving these responses contributes to the
pathogenesis of mental disorders such as depression and
post-traumatic stress disorder (McEwen, 2003). This
association between chronic exposure to stress and the
induction of mental disorders provides an opportunity to
study neural mechanisms contributing to their etiology.
Furthermore, animal models such as the chronic mild or
unpredictable stress model of depression have proved to be
valuable tools in understanding the neurobiological rela-
tionship between stress and depression (Willner, 1997). For
instance, many of the biochemical sequelae that are seen in
some depressed patients are also seen in animals subjected
to chronic unpredictable stress (CUS; Lopez et al, 1998).
In addition to increased adrenal steroid secretion, these
include increased cortical 5-HT2Areceptors (Ossowska et al,
2001), decreased hippocampal 5-HT1A receptors (Lopez
et al, 1998), downregulated glucocorticoid receptors (Froger
et al, 2004), and increased levels of peripheral and central
pro-inflammatory molecules (Grippo et al, 2003). These
findings demonstrate that at least on a physiological level,
the chronic unpredictable stress model is a reliable tool in
examining possible biochemical changes underlying de-
One neuroanatomical region sensitive to stress is the
hippocampus, a region also highly associated with both
affective diseases such as depression (McEwen, 2003) and
with basic cognitive functioning and the regulation of
learning and memory (McEwen, 2001). The hippocampus is
very susceptible to stress-induced morphological and
electrophysiological alterations. In particular, decrements
in long-term potentiation (Alfarez et al, 2003; Pavlides et al,
2002) and dendritic atrophy and de-branching (Galea et al,
1997; Vyas et al, 2002) have been observed following long-
term stress exposure. These physiological changes often
correlate well with stress-induced impairments in learning,
memory, and reversal learning in animals (Francis et al,
Online publication: 27 September 2004 at http://www.acnp.org/citations/
Received 3 May 2004; revised 9 September 2004; accepted 22
*Correspondence: Dr BB Gorzalka, Department of Psychology,
University of British Columbia, 2136 West Mall, Vancouver, BC,
Canada V6T 1Z4, Tel: þ1 604 822 3095, Fax: þ1 604 822 6923,
E-mail: firstname.lastname@example.org and Dr CJ Hillard, Department of
Pharmacology and Toxicology, Medical College of Wisconsin, 8701
Watertown Plank Road, Milwaukee, WI 53226, USA, Tel: þ1 414 456
8493, Fax: þ1 414 456 6545, E-mail: email@example.com
4These authors contributed equally to this manuscript.
Neuropsychopharmacology (2005) 30, 508–515
& 2005 Nature Publishing GroupAll rights reserved 0893-133X/05 $30.00
1995; de Quervain et al, 1998; Luine et al, 1994; Vascon-
cellos et al, 2003).
Recent evidence has accumulated suggesting that there
are functional interactions between the endogenous canna-
binoid (eCB) system and stress circuitry (Hill and Gorzalka,
2004; Patel et al, 2004), and furthermore that the eCB system
is involved in both emotional regulation (Martin et al, 2002)
and synaptic transmission in the hippocampus (Carlson
et al, 2002). The association of eCB signaling and stress-
related mental disorders may occur at multiple loci
throughout the brain. However, the cannabinoid type 1
(CB1) receptor is found in abundance in the hippocampus
(Herkenham et al, 1991), which, as previously mentioned,
appears to be an important structure in the functional
neuroanatomy of depression (McEwen, 2003). This recep-
tor, which is located primarily on GABAergic interneurons
within the hippocampus (Irving et al, 2000; Katona et al,
1999), negatively regulates adenylyl cyclase and calcium
channels via coupling to Gi/o subunits, thus reducing
neurotransmitter release. The CB1receptor is responsive
to exogenous cannabinoids, such as tetrahydrocannabinol
(THC) from cannabis, as well as putative endogenous
ligands, including 2-arachidonylglycerol (2-AG; Sugiura
et al, 1995), which has shown to be formed in response to
increased hippocampal neuronal activity (Stella et al, 1997).
Hippocampal CB1 receptor activation, like stress, is
associated with alterations in various cognitive tasks
(Lichtman et al, 2002; Hampson and Deadwyler, 1998,
1999). In humans, cannabis consumption is known to cause
impairments in learning, retention and memory retrieval
(Pope et al, 2001; Solowij et al, 2002), an effect that has also
been documented in animals treated with exogenous CB1
receptor ligands (Hampson and Deadwyler, 1998; Chaperon
and Thiebot, 1999). These parallels suggest the possibility
that eCB signaling in the hippocampus could play a
functional role in the cognitive deficits seen following
The limbic forebrain is another neuroantomical region
where convergence of stress and endocannabinoid signaling
could be functionally significant. Structures in the limbic
forebrain, such as the prefrontal cortex, are very sensitive to
stress exposure (Mizoguchi et al, 2000) and are believed to
mediate many higher order cognitive abilities such as
behavioral flexibility (Miller, 2000). Like the hippocampus,
limbic forebrain neurons express CB1receptors, albeit to a
lesser degree than the hippocampus (Herkenham et al,
1991). The functional role of receptors located in this region
is even less well known than that of the receptors in the
hippocampus. However, deficits in CB1receptor signaling,
as revealed through mice deficient in the CB1receptor gene,
result in specific impairments in cognitive flexibility as
manifested through increased perseveratory behaviors
(Varvel and Lichtman, 2002). Since the limbic forebrain is
believed to regulate flexibility and decision making
processes, it is possible that CB1receptors located in this
region play a functional role in behavioral inhibition and
Mice deficient in the CB1 receptor gene also exhibit
enhanced susceptibility to the anhedonic effects of chronic
variable stress (Martin et al, 2002), suggesting that CB1
receptor activity may counteract or suppress some of the
negative affective or cognitive effects induced by chronic
exposure to unpredictable stress. Due to the ability of both
chronic stress exposure and CB1 receptor activity to
modulate cognitive function we tested the hypothesis that
alterations in eCB signaling contribute to the cognitive
impairments induced by chronic stress. Our data indicate
that chronic, nonhabituating stress results in a decrease in
functional eCB signaling within the hippocampus without
affecting eCB activity in the limbic forebrain, and that the
stress-induced impairment in reversal learning can be
reversed by exogenous activation of CB1receptors. These
data suggest that pharmacological modulation of eCB
signaling could represent a novel approach to the treatment
of cognitive deficits that accompany a variety of anxiety-
related neuropsychiatric disorders.
MATERIALS AND METHODS
Seventy-day-old male Long-Evans rats (300g) housed in
groups of three in triple mesh wire caging were used in
this study. Colony rooms were maintained at 211C, and
on a reverse 12h light/dark cycle, with lights off at 0900.
All rats were given ad libitum access to Purina Rat Chow
and tap water, except during deprivation periods of the
stressing protocol (outlined below). Subjects were randomly
assigned into two groups and were either subjected to 21
days of CUS (2–3 stressors a day from the following list:
30min tube restraint; 30min exposure to white noise/
stroboscopic illumination; 5min forced swim; 18h food
and/or water deprivation; 3h cage rotation; 18h social
isolation), or acted as cage controls and were handled four
times weekly. This chronic unpredictable stress paradigm
has been successfully utilized in our laboratory (Brotto et al,
2001) to examine the effects of stress on copulatory
behavior and is adapted from the chronic mild stress
paradigm (see Willner, 1997). All treatments of animals
were approved by the Canadian Council for Animal Care
and the standards of the Animal Ethics Committee of the
University of British Columbia. Testing groups were divided
into subsets that were used for either behavioral analysis
(n¼9 or 10) or for biochemical analyses (n¼4–6). Animals
used for all biochemical assays were rapidly decapitated in
the morning after the 21st day of stress exposure following
12h of overnight social isolation. Brains were removed and
the hippocampus and limbic forebrain (all tissue rostral to
the amygdala, excluding the olfactory bulbs) were sectioned
and frozen in liquid nitrogen within 5min of decapitation
and stored at ?801C until analysis. Trunk blood was also
collected upon decapitation for measurement of plasma
Tissue Preparation for Endocannabinoid Quantification
Brain tissue samples were subjected to a lipid extraction
process exactly as described previously (Patel et al, 2003).
The content of both 2-AG and the other major eCB ligand,
anandamide (AEA; Devane et al, 1992) within lipid extracts
were determined using isotope-dilution liquid chromato-
graphy/mass spectrometry as described previously (Patel
et al, 2003).
Hippocampal endocannabinoids and stress
MN Hill et al
CB1Receptor Binding Assays and Western Blots
To make membranes, dissected brain sections were homo-
genized in 5ml TME buffer (50mM Tris-HCl, 1mM EDTA,
3mM MgCl2, pH 7.4 with Tris base) using a Dounce
homogenizer. Membranes were centrifuged at 17500g for
20min, and the resulting pellet was resuspended by
homogenizing in 2–2.5ml TME bufferþ1mM sodium
orthovanadate. Protein concentrations were determined by
Bradford method (Bio-Rad, Hercules, CA).
CB1 receptor binding assays were performed using a
Multiscreen Filtration System with Durapore 1.2-mM filters
(Millipore, Bedford, MA, USA). Incubations (total volu-
me¼0.2ml) were performed with TME buffer containing
1mg/ml bovine serum albumin (TME/BSA). Membranes
(10mg protein per incubate) were added to the wells
containing 0.25, 0.5, 1.0, or 2.5nM3H-CP 55940. In total,
10mM D9-THC was used to determine nonspecific binding.
For Western blotting procedures, all membranes were
made to a 3mg/ml final concentration in TME buffer.
Laemmli loading buffer (4?) was added to each sample,
and samples denatured at 651C for 5min. Protein samples
were loaded onto a 10% SDS-PAGE gel, separated by
electrophoresis, and transferred onto a nitrocellulose
membrane. Nonspecific membrane binding was blocked at
41C with an overnight incubation in phosphate-buffered
saline containing 0.2% Tween-20 (PBST) and 2% milk.
Primary anti-rCB1 antibody was diluted 1:300 in PBST/milk,
and incubated with the membrane overnight at 41C. After
washing, the membrane was incubated with HRP-conju-
gated goat anti-rabbit secondary antibody (diluted 1:3000 in
PBST/milk) for 45min at room temperature.
Upon collection of trunk blood, samples were stored
overnight at 41C and centrifuged the following morning at
10g for 10min. Plasma was removed and centrifuged again
at 10g for 10min. Bound plus free serum corticosterone was
measured through radioimmunoassay, using a previously
validated method (Weinberg and Bezio, 1987). Antiserum
was obtained from Immunocorp Montreal, Canada and
tracer was obtained from Mandel Scientific, Guelph,
Canada. Dextran coated charcoal was used to absorb and
precipitate free steroids after incubation.
The Morris water maze (MWM) used in this experiment was
a large, circular pool (193cm diameter) that was filled to a
depth of 70cm with water (221C) that was made opaque
through the addition of nontoxic white paint (Washable Dry
Temp, Palmer Paint Products, Troy, MI, USA). The
platform was a cylindrical jar (17?8.5cm2) that had a
square wire mesh platform on top that was submerged 3cm
below the surface of the water. Distinctive distal visual cues
surrounded the pool and remained in place for the duration
of the experiment. A computer-based automated tracking
system (HVS Image, Hampton, UK) was used to calculate
the swim speed and escape latencies (the time each subject
required to locate the hidden platform after being released)
of each subject.
Testing in the MWM began on day 16 of the stress regimen.
All testing in the MWM occurred in the morning prior to the
induction of any stressors that day. Animals were trained in a
standard protocol for acquisition and reversal learning that
has been previously described (Varvel and Lichtman, 2002).
Briefly, rats were trained with five acquisition sessions that
consisted of four trials per day with an intertrial interval of
30s. During the acquisition period, the hidden platform
remained in the same fixed position, which was randomly
determined for each rat. On day 22, after five sessions of
acquisition training, all rats were subjected to a reversal test
in which the platform was moved to the opposite side of the
tank, but all other testing factors remained constant. For
reversal testing, subjects in both the stress and control groups
were divided up into two subgroups: administration of
vehicle (1:1:18 Tween 80: dimethyl sulfoxide/saline) or 10mg/
kg HU-210 (Tocris-Cookson, Bristol, UK). Subjects received
an injection of either vehicle or HU-210 30min prior to the
first of four reversal trials. As in the acquisition task, all rats
were released from the same point, but the platform was
moved to the opposite quadrant of the pool. Escape latencies
and amount of time spent in each quadrant of the pool were
Comparison of the effects of CUS on CB1receptor binding
and protein expression, as well as on eCB synthesis and
plasma corticosterone values, were carried out using an
independent t-test. Behavioral data in the MWM was
analyzed using a two-factor analysis of variance, and post
hoc analysis performed using a Tukey HSD test. Significance
was established against an alpha level of 0.05.
Biochemical Changes in the eCB System Induced by
Animals exposed to 21 days of CUS exhibited increased
po0.05) when compared to nonstressed animals (stressed
animals: 17.373.4dg/ml; control animals: 3.471.1dg/ml;
mean7SEM), indicating that the paradigm used elicited a
nonhabituating stress response. Following CUS, rats ex-
hibited a 50% reduction in CB1receptor protein expression
in the hippocampus (t(9)¼2.5, po0.05) compared to
nonstressed controls (Figure 1a). In addition, stressed
animals exhibited a 40% reduction in the Bmax of CB1
receptor binding (t(22)¼3.1, po0.01) without a change in
the KDof the receptor for [3H]CP55940 (t(22)¼1.2, NS)
(Table 1). In the limbic forebrain, no differences in CB1
receptor protein (t(6)¼0.4, NS), Bmax(t(22)¼0.3, NS) or
KD for [3H]CP55940 (t(22)¼0.1, NS) were observed
between CUS-treated rats and cage controls (Figure 1b
and Table 1).
Stressed animals exhibited a 40% reduction in 2-AG
content (t(7)¼4.9, po0.005), AEA content was not
significantly altered (t(8)¼2.0, NS) (Figure 2a). In the
limbic forebrain, there were no differences in 2-AG
(t(8)¼0.3, NS) or AEA (t(8)¼1.2, NS) content between
CUS-exposed rats and cage controls (Figure 2b).
Hippocampal endocannabinoids and stress
MN Hill et al
Behavioral Changes Induced by Chronic Stress and
Their Reversal Following Cannabinoid Agonist
Behavioral testing in the MWM task began after 16 days of
exposure to CUS. Stressed animals exhibited no cognitive
deficits during the acquisition phase of the task in which the
animals learned the location of a platform that provided an
escape from the water (F(1,6)¼0.3, NS). Daily escape
latency times during acquisition training can be viewed in
Figure 3a. However, during reversal trials, in which the
platform was placed on the opposite quadrant of the water
maze, stressed animals exhibited a deficit in learning the
location of the new platform (F(3,34)¼2.4, po0.05). This
deficit was not accompanied by any significant changes in
swim speed (F(3,34)¼1.8, NS) (data not shown). Post hoc
expression as determined through Western blot analysis in the
hippocampus (a) and limbic forebrain (b). Values are denoted as
means7SEM of band density (in arbitrary units). Representative Western
blot pairs are seen next to the graphs. *Significantly different from control
Effect of 21 days of chronic stress on CB1receptor protein
Table 1 The Effect of Chronic Stress on CB1Receptor Binding in
the Hippocampus and Limbic Forebrain
Exposure to 21 days of chronic stress resulted in diminished [3H]CP55940
binding to CB1receptor in the hippocampus, while having no effect on CB1
receptor binding in the limbic forebrain. Since the effect is limited to the Bmax,
this suggests that reduction in binding is due to a reduction in the size of the
available receptor pool and not due to changes in affinity of the receptor itself.
This idea is complimented by the protein expression for the CB1receptor
(Figure 1), which demonstrates that expression of this receptor is reduced to
the same degree as the binding. Data are presented as mean values7SEM.
Significantly different values (po0.05) denoted by *.
(ng/g tissue weight)
(µg/g tissue weight)
(µg/g tissue weight)
* * * *
(ng/g tissue weight)
control (po0.05) (n¼5/group).
Effect of 21 days of chronic stress on AEA and 2-AG content in the hippocampus (a) and limbic forebrain (b). *Significantly different from
Hippocampal endocannabinoids and stress
MN Hill et al
analysis indicated that there were no significant differences
in escape latency in the first two trials of reversal learning,
but on the third and fourth trials the stressed animals were
significantly impaired compared to the nonstressed group
(po0.05). Analysis of the percent time spent in each
quadrant during the reversal trials indicated that the escape
latency deficit seen in the stressed animals was paralleled by
an increased percentage of time spent in the quadrant in
(F(3,34)¼3.4, po0.01). Post hoc analysis revealed that
stressed animals spent significantly more time in the trained
quadrant in trials 3 and 4 (po0.05). Exogenous adminis-
tration of 10mg/kg of the CB1receptor agonist HU-210,
prior to the reversal task, completely blocked the deficits in
reversal learning and perseveratory behavior seen following
CUS (po0.05; Figure 3b and c).
These experiments demonstrate that exposure of rats to
CUS for 21 days is associated with robust reductions in both
2-AG content and CB1 receptor density within the
hippocampus, and the induction of perseveratory behavior
in the MWM. Rats that had been subjected to CUS exhibited
difficulties in learning a new platform location when it was
shifted to the opposite side of the maze. This increase in
escape latency was paralleled by an increase in the percent
of time spent in the initial training quadrant, indicating that
the stressed animals were perseverating. The cognitive
impairments induced by CUS were strikingly similar to
those observed in CB1
ments in extinction and perseveratory behavior in a variety
of cognitive tasks including the MWM (Varvel and Licht-
man, 2002), suggesting that the cognitive impairments
induced by chronic stress could be a consequence of
deficient eCB signaling. This enhanced perseveratory
behavior was attenuated by pharmacological enhancement
of CB1 receptor activity, consistent with mediation by
deficient eCB signaling. This suggests that a physiological
response to CUS is a significant attenuation of eCB signaling
in the hippocampus, which in turn could contribute to the
cognitive impairments induced by chronic stress. However,
this suggestion remains speculative until it is verified
through pharmacological antagonism of the eCB system and
other experiments are carried out to address the causal
nature of the relationship. Chronic stress was found to have
no effect on either CB1receptor binding or synthesis of the
endogenous ligands in the limbic forebrain, another region
that is highly activated during environmental stress (Bubser
and Deutch, 1999).
In the present studies, exposure to CUS did not influence
acquisition of learning the MWM. This finding was
surprising, as exposure to chronic stressors is typically
shown to impair spatial learning (Luine et al, 1994).
However, many of these studies utilized chronic, homotypic
stress paradigms (eg chronic restraint), which are known to
induce dendritic atrophy in the hippocampus not seen in
animals exposed to a variable stress paradigm such as the
one utilized here (Vyas et al, 2002). However, it should be
noted that there are discrepancies in the literature on the
effects of chronic stress on spatial learning. While many
?/?mice, which also show impair-
studies suggest a decrement occurs (Vasconcellos et al,
2003; Touyarot et al, 2004; Luine et al, 1994), others suggest
that there may be an enhancement under some conditions
(Bartolomucci et al, 2002). Clearly, the mechanisms
contributing to the effects of stress on cognitive functioning
require further investigation.
Escape Latency (s)
Percent Time Spent in Initial Training
day 5day 4day 3day 2day 1
Escape Latency (s)
treatment on acquisition and reversal tasks in the Morris water maze. (a)
CUS exposure did not have an effect on escape latency during platform
acquisition in the MWM (n¼18/group). (b) Chronic stress (S) impaired
reversal learning in the MWM as relative to nonstressed (NS) animals.
Pretreatment of rats with 10mg/kg HU-210 (CB), a selective and potent
CB1receptor agonist, prevented the deficit in reversal learning seen in
vehicle-treated (V) stressed animals (V) (n¼9 or 10/group). (c) Stressed
animals were found to spend a higher proportion of time in the training
quadrant, an effect that was attenuated by pretreatment with HU-210.
*Significantly different (po0.05) from all other groups (n¼9 or 10/group).
Effect of chronic stress and subsequent exogenous cannabinoid
Hippocampal endocannabinoids and stress
MN Hill et al
It is interesting that chronic stress resulted in both
downregulation of CB1receptors and a decrease in 2-AG
content in the hippocampus. Several studies have demon-
strated that chronic treatment of rats (Breivogel et al, 1999)
and mice (Sim-Selley and Martin, 2002) with CB1receptor
agonists results in downregulation of the CB1 receptor.
However, these studies were conducted using high doses of
agonists with long biological half-lives. In contrast, Romero
et al (1995) demonstrated that i.p. administration of
anandamide once daily for 5 days resulted in a significant
increase in CB1receptor density in the hippocampus with
no effect in the limbic forebrain. Thus, these studies provide
evidence that moderate activation of CB1receptor activity
can result in its upregulation in the hippocampus. By
analogy, it is possible that the stress-induced decrease in
hippocampal eCB content drives the decrease in CB1
receptor density due to withdrawal of a trophic factor for
CB1receptor expression, in this case, its ligand.
Alternatively, the change in CB1receptor density and eCB
content could occur independently, both driven by the
physiologic changes that accompany stress but not depen-
dent on each other. Interestingly, it has been suggested that
glucocorticoids (GC) exert negative regulation over CB1
receptor transcription (Mailleux and Vanderhaeghen, 1993).
This idea was supported here as this stress protocol induced
both elevated GC levels and reduced hippocampal CB1
receptor density. However, the lack of downregulation of
CB1 receptors in the limbic forebrain suggests that the
mechanisms involved in regulation of CB1receptor density
in the hippocampus must include more than GC changes. Di
et al (2003) have recently shown that GCs stimulate eCB
synthesis through a nongenomic, fast-acting mechanism,
which contrasts with the present findings in the hippocam-
pus and suggests that the modulation of eCB content by
chronic exposure to unpredictable stress is not solely GC
mediated and likely brain region specific.
It is possible that increased leptin levels could be driving
the decrease in eCB content in the hippocampus seen in this
study. The satiety peptide leptin is known to decrease brain
eCB content (Di Marzo et al, 2001). Since leptin production
is elevated following chronic unpredictable stress (Gamaro
et al, 2003), it is possible that increased leptin levels could
be driving the decrease in eCB content in the hippocampus
seen in this study. It is interesting in light of this argument
that rats given free access to a highly palatable food for 10
weeks, which significantly elevates plasma leptin concentra-
tions, exhibit a 30–50% decrease in CB1receptor density in
the hippocampus (Harrold et al, 2002). It is also possible
that changes in other neurotransmitter systems could be
mediating the changes in eCB levels; however, due to the
fact that stress stimulates excitatory transmission in the
hippocampus (McEwen and Magarinos, 1997), one would
actually expect an increase and not a decrease in eCB levels
(Stella et al, 1997). At present, it is difficult to speculate on
the exact mechanism of action of these physiological
changes, given that the biosynthetic pathways of eCB
synthesis are not well understood.
A growing body of evidence suggests that eCB activity
plays a crucial role in cognitive processes that require
inhibition of a previously learned behavior, such as
extinction or reversal learning. Genetic deletion of the CB1
receptor gene results in animals that exhibit perseveration
during reversal learning (Varvel and Lichtman, 2002) or an
inability to extinguish aversive memories in fear condition-
ing paradigms (Marsicano et al, 2002). This study comple-
ments these previous data by showing that deficits in
flexibility are apparent when eCB signaling is blunted in the
hippocampus, suggesting that deficits in eCB signaling in
areas of the limbic forebrain, such as the prefrontal cortex,
are not required for inhibition of a behavioral strategy. This
does not preclude the idea that forebrain structures play a
fundamental role in the manifestation of cognitive flex-
ibility, but suggests that the ability of eCB signaling to affect
this behavior is likely mediated through its actions in the
hippocampus. It is known that glutamatergic projections
originating from the ventral subiculum of the hippocampus
terminate in the prelimbic region of the prefrontal cortex
(PFC; Carr and Seasack, 1996), forming a connection which
could subserve such an interaction. Given that CB1receptor
activation inhibits hippocampal GABA release (Katona et al,
1999), our data suggest that CUS could increase local
GABAergic tone and thus decrease the activity of this
hippocampal–PFC pathway. Thus, modulation of prefrontal
cortical activity could ultimately mediate the disturbance in
higher order cognitive tasks that was seen here; however,
this hypothesis requires further investigation.
Aside from demonstrating that deficits in cognitive
flexibility are associated with attenuated endocannabinoid
signaling in the hippocampus, this study also demonstrates
that these behavioral changes, previously only attained
through genetic manipulation of mice or pharmacological
blockade of the CB1receptor (Varvel and Lichtman, 2002;
Marsicano et al, 2002), can be induced through environ-
mental manipulations. Recently, it was shown that enriched
environmental exposure results in 10-fold elevations in
hippocampal eCB content (Wolf and Matzinger, 2003),
which together with the data presented here suggest that
hippocampal eCB signaling is vulnerable to environmental
changes, becoming blunted during periods of prolonged
stress and elevated following enrichment. Owing to the
association between affective disease and stress, and the fact
that the CUS paradigms have been shown to model many of
the neurochemical disturbances seen in depression (Lopez
et al, 1998; Ossowska et al, 2001; Willner, 1997), these
findings also suggest that the eCB system may be another
system that could be disturbed in depression, and thus play
a role in the manifestation of some of the symptoms of
Several research groups have suggested that eCB signaling
in the hippocampus plays a functional role in the forgetting
process (Terranova et al, 1996; Hampson and Deadwyler,
1998; Varvel and Lichtman, 2002), an idea supported by our
findings that blunted hippocampal eCB signaling was
associated with deficits in behavioral flexibility. Interest-
ingly, many stress-related mental diseases, such as post-
traumatic stress disorder and depression, are characterized
by problems with forgetting and behavioral flexibility that
are manifested as pathological tendencies to ruminate or
perseverate on negative information (Bagby et al, 1999;
Gold and Chrousos, 2002; Heresco-Levy et al, 2002; King,
2002). Considering the present evidence and previous
evidence from other laboratories, one may postulate that
stress-induced deficits in cognitive flexibility are mediated
in part by stress-induced reductions in hippocampal eCB
Hippocampal endocannabinoids and stress
MN Hill et al
signaling. If stress-induced attenuation of eCB signaling is
involved in the manifestation of perseveratory and rumi-
natory behavior, then pharmacological manipulation of this
system could prove to be a novel approach to treatment of
these cognitive symptoms.
This research was supported by NIH Grant R01-DA016967
and an Independent Investigator Award from NARSAD to
CJH; a Natural Sciences and Engineering Research Council
(NSERC) grant to BBG; an NSERC postgraduate scholarship
and a Michael Smith Foundation for Health Research
(MSFHR) Research Training Award to MNH; an NIH-NRSA
grant (F30 DA15575) to SP; an NIH-NRSA grant (F32
DA16510) to DJR. We like to thank Maric Tse, Indy Gill, Eda
Karacabeyli and Wayne Yu for their technical assistance.
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